Synaptic dysfunction in human immunodeficiency virus type-1-positive subjects: inflammation or impaired neuronal plasticity?

Many people infected with the human immunodeficiency virus type-1 (HIV) exhibit mild or severe neurological problems, termed HIV-associated neurocognitive disorder (HAND), even when receiving antiretroviral therapy. Thus, novel adjunctive therapies must be developed to overcome the neurotoxic effect of HIV. New therapies require a better understanding of the molecular and cellular mechanisms of HIV-induced neurotoxicity and the risk factors that, besides inflammation and T-cell depletion and drugs of abuse, render the central nervous system (CNS) a target of HIV-induced neurotoxicity. HIV appears to impair neuronal plasticity, which refers to the innate ability of the CNS respond to injury and promote recovery of function. The availability of brain-derived neurotrophic factor (BDNF), a potent neurotrophic factor that is present in abundance in the adult brain, is essential for neuronal plasticity. BDNF acts through a receptor system composed of Trk and p75NTR. Here, we present experimental evidence that some of the clinical features of HIV-mediated neurological impairment could result from altered BDNF/TrkB/p75NTR regulation and function.

Brain-derived neurotrophic factor as a prototype neuroprotective factor against HIV-1-associated neuronal degeneration.

Patients with human immunodeficiency virus type 1 (HIV-1) infection develop a broad spectrum of motor impairments and cognitive deficits, which follow or parallel cellular loss and atrophy in their brains. The viral envelope glycoprotein 120 (gp120) has been suggested to be a causal agent of neuronal loss. Therefore, reducing gp120 neurotoxicity may prevent neuronal degeneration seen in these patients. Here, we describe in vitro and in vivo experimental evidence that gp120 toxicity can be reduced by brain-derived neurotrophic factor (BDNF), a naturally occurring peptide that has been shown to block neurotoxin and trauma-induced neuronal injury. Moreover, we review the survival promoting properties of BDNF and the issues concerning its delivery into the brain, in an attempt to explain the rationale for exploring BDNF as a prototype trophic factor for a therapy to reduce neuronal cell death in HIV-1 infected patients.

Exogenous gangliosides, neuronal plasticity and repair, and the neurotrophins.

Gangliosides, a heterogeneous family of glycosphingolipids abundant in the brain, have been shown to affect neuronal plasticity during development, adulthood and aging. This review will examine old and recent evidence that exogenous gangliosides and in particular GM1, the prototype member of this family, exhibit multimodal neurotrophic effects. Since these compounds are a potential therapeutic tool for the treatment of various forms of acute or chronic neurodegenerative diseases, understanding the dynamic interplay of gangliosides and neuronal cells is essential in the effort to cure neurological disorders. Focus will be given to the novel and provocative hypothesis that gangliosides' neuroprotective properties may derive from their ability to mimic endogenous neurotrophic factors.

TrkA induces differentiation but not apoptosis in C6-2B glioma cells.

Nerve growth factor (NGF) binds to the TrkA tyrosine kinase and the p75 neurotrophin receptors. Depending upon which receptor is activated, NGF can induce differentiation or apoptosis. C6-2B glioma cells express the p75 receptor, but NGF decreases their growth only when TrkA is introduced (C6trk). It is unclear, however, whether TrkA reduces C6-2B cell growth by apoptosis or differentiation. To examine which mechanisms account for the anti-proliferative effect of NGF in these cells, we first analyzed whether NGF causes apoptosis by flow cytometry, two-site immunoassay and in situ TUNEL. None of these methods indicated that C6trk undergo apoptosis. Additional apoptotic markers, such as Bcl-2, Bax, Bad, p53, caspase 3, and NF-kappaB were also used. C6trk cells exhibited lower levels of Bcl-2 compared with the parental C6 mock cells, but no changes in the levels of other apoptotic proteins. Moreover, NGF increased AP-1 binding activity in C6trk cells, suggesting that NGF may induce differentiation. We then examined whether TrkA changes the glioma phenotype. In C6trk cells, but not in C6mock cells, NGF enhanced the levels of neuron-specific enolase as well as the levels of A2B5 and 2', 3'-cyclic nucleotide 3'-phosphodiesterase, markers for oligodendrocytes, without affecting the expression of other neuronal markers. Our data suggest that the antiproliferative properties of TrkA may rely on its ability to induce differentiation of C6 cells from undifferentiated glioma to oligodendrocytes.

Interleukin-10 prevents glutamate-mediated cerebellar granule cell death by blocking caspase-3-like activity.

Interleukin-10 (IL-10) has been shown to reduce neuronal degeneration after CNS injury. However, the molecular mechanisms underlying the neuroprotective properties of this cytokine are still under investigation. Glutamate exacerbates secondary injury caused by trauma. Thus, we examined whether IL-10 prevents glutamate-mediated cell death. We used rat cerebellar granule cells in culture because these neurons undergo apoptosis upon exposure to toxic concentrations of glutamate (100-500 microm) or NMDA (300 microm). Pretreatment of cerebellar granule cells with IL-10 (1-50 ng/ml) elicited a dose- and time-dependent reduction of glutamate-induced excitotoxicity. Most importantly, IL-10 reduced the number of apoptotic cells when added to the cultures together or 1 hr after glutamate. Using patch-clamping and fluorescence Ca(2+) imaging techniques, we examined whether IL-10 prevents glutamate toxicity by blocking the function of NMDA channel. IL-10 failed to affect NMDA channel properties and to reduce NMDA-mediated rise in intracellular Ca(2+). Thus, this cytokine appears to prevent glutamate toxicity by a mechanism unrelated to a blockade of NMDA receptor function. Various proteases, such as caspase-3, and transcription factors, such as nuclear factor kappaB (NF-kappaB), have been proposed to participate in glutamate-mediated apoptosis. Thus, we examined whether IL-10 modulates the activity of these apoptotic markers. IL-10 blocked both the glutamate-mediated induction of caspase-3 as well as NF-kappaB DNA binding activity, suggesting that the neuroprotective properties of IL-10 may rely on its ability to block the activity of proapoptotic proteins.

Dexamethasone reduces the expression of p75 neurotrophin receptor and apoptosis in contused spinal cord.

Apoptosis is an important cause of secondary cell death in spinal cord injury (SCI). SCI induces the expression of the low affinity neurotrophin receptor p75 (p75NTR), that in the absence of the high affinity component, TrkA, can promote cell death by apoptosis. We therefore hypothesized that a reduction of p75NTR expression in SCI may increase tissue sparing and therefore improve recovery of function. As a tool to test our hypothesis we used the synthetic glucocorticoid dexamethasone (DEX) to down-regulate p75NTR expression. A standardized thoracic spinal cord contusion injury was produced in female rats. Laminectomized and SCI rats received various doses of DEX immediately after injury and the treatment was continued daily for 7 days. DEX, given at high doses (20 mg/kg, s.c.) but not at low doses (1 or 8 mg/kg) prevented the increase in p75NTR mRNA and protein in SCI rats, without affecting the expression of TrkA. High doses of DEX also reduced cellular apoptosis both in white and gray matters. This effect correlated with the ability of DEX to accelerate behavioral recovery of function measured by a combined behavioral score. These data suggest that reduction of p75NTR in SCI may be a therapeutic strategy to limit cell and tissue damage and therefore to improve recovery of function in SCI patients.

Dexamethasone induces TrkA and p75NTR immunoreactivity in the cerebral cortex and hippocampus.

Nerve growth factor (NGF) plays a crucial role in synaptic plasticity during brain development and adulthood by activating a dual receptor system composed of TrkA and p75 (p75NTR) receptors. Exogenous NGF modulates the expression of both receptors. Little is known about the ability of endogenous NGF to regulate the expression of these receptors in basal forebrain cholinergic terminals. The ability of glucocorticoids to increase NGF expression in the hippocampus prompted us to investigate whether the synthetic glucocorticoid dexamethasone (DEX) increases TrkA and p75NTR expression in NGF-target cholinergic neurons in developing rats. We first examined the effect of DEX on NGF mRNA by in situ hybridization. DEX given systemically (0.5 mg/kg, sc) for 1 week to 7-day-old rats elicited an increase in NGF mRNA levels in the dentate gyrus of the hippocampus and superficial layers II and III of the cerebral cortex. Immunohistochemical analysis of p75NTR and TrkA levels revealed a dramatic increase in p75NTR immunoreactivity (IR) in both basal forebrain and hippocampus and TrkA IR in the hippocampus. Interestingly, in DEX-treated rats more axonal terminals were immunopositive for p75NTR in the hippocampus and cortex, suggesting an increase in p75NTR IR in cell bodies as well as in terminals. Our data indicate that the endogenously produced NGF elicits biological changes similar to those of the exogenously delivered NGF. We suggest that glucocorticoids might regulate and coordinate cholinergic neuronal maturation by increasing the biosynthesis of NGF.

Basic fibroblast growth factor increases long-term survival of spinal motor neurons and improves respiratory function after experimental spinal cord injury.

Acute focal injection of basic fibroblast growth factor (FGF2) protects ventral horn (VH) neurons from death after experimental contusive spinal cord injury (SCI) at T8. Because these neurons innervate respiratory muscles, we hypothesized that respiratory deficits resulting from SCI would be attenuated by FGF2 treatment. To test this hypothesis we used a head-out plethysmograph system to evaluate respiratory parameters in conscious rats before and at 24 hr and 7, 28, and 35 d after SCI. Two groups of rats (n = 8 per group) received either FGF2 (3 microg) beginning 5 min after injury or vehicle (VEH) solution alone. We found significantly increased respiratory rate and decreased tidal volume at 24 hr and 7 d after SCI in the VEH-treated group. Ventilatory response to breathing 5 or 7% CO(2) was also significantly reduced. Recovery took place over time. Respiration remained normal in the FGF2-treated group. At 35 d after injury, histological analyses were used to compare long-term neuron survival. FGF2 treatment doubled the survival of VH neurons adjacent to the injury site. Because the number of surviving VH neurons rostral to the injury epicenter was significantly correlated to the ventilatory response to CO(2), it is likely that the absence of respiratory deficits in FGF2-treated rats was caused by its neuroprotective effect. Our results demonstrate that FGF2 treatment prevents the respiratory deficits produced by thoracic SCI. Because FGF2 also reduced the loss of preganglionic sympathetic motoneurons after injury, this neurotrophic factor may have broad therapeutic potential for SCI.

Dexamethasone induces hypertrophy of developing medial septum cholinergic neurons: potential role of nerve growth factor.

Glucocorticoid hormones influence neuronal plasticity during development; however little is known about the mechanisms of this trophic activity. Because glucocorticoids increase nerve growth factor (NGF) synthesis in selected brain areas and NGF plays a role in the development of basal forebrain cholinergic neurons, we tested the hypothesis that glucocorticoids may foster maturation of the cholinergic phenotype during postnatal development via the induction of NGF biosynthesis. The synthetic glucocorticoid dexamethasone (DEX) was injected systemically (0.5 mg/kg, s.c.) once a day for 1 week in 7-d-old (P7) rats. DEX elicited an increase in NGF mRNA and protein levels in the cerebral cortex and hippocampus as well as specific NGF responses, such as TrkA tyrosine phosphorylation in the septum, choline acetyltransferase (ChAT) and p75 neurotrophin receptor (p75NTR) immunoreactivity, and a relative number of cholinergic neurons in the medial septum. To examine whether the effect of DEX is age-related, we treated 1- and 14-d-old rats with DEX for 1 week. DEX increased NGF expression in rats treated from P1 to P8 but not in those treated from P14 to P21. The age-related increased expression of NGF correlated with the induction of ChAT immunoreactivity in the medial septum. Moreover, in the spinal cord, neither NGF nor ChAT levels were increased by DEX, suggesting that the glucocorticoid-mediated changes seen in the basal forebrain are associated with specific NGF responses. Our data suggest that by increasing NGF levels, glucocorticoids may play a role in the maturation of postnatal cholinergic neurons.

Activity-dependent release of brain-derived neurotrophic factor underlies the neuroprotective effect of N-methyl-D-aspartate.

The molecular mechanism(s) of N-methyl-D-aspartate (NMDA) neuroprotective properties were investigated in primary cultures of cerebellar granule cell neurons. Granule cells express the neurotrophin receptor TrkB but not TrkA or TrkC. In these cells, the TrkB ligand brain-derived neurotrophic factor (BDNF) prevents glutamate toxicity. Therefore, we have tested the hypothesis that NMDA activates synthesis and release of BDNF, which may prevent glutamate toxicity by an autocrine loop. Exposure of granule cells for 2 and 5 min to a subtoxic concentration of NMDA (100 microM) evoked an accumulation of BDNF in the medium without concomitant changes in the intracellular levels of BDNF protein or mRNA. The increase in BDNF in the medium is followed by enhanced TrkB tyrosine phosphorylation, suggesting that NMDA increases the release of BDNF and therefore the activity of TrkB receptors. To examine whether BDNF and TrkB signaling play a role in the NMDA-mediated neuroprotective properties, neurons were exposed to soluble trkB receptor-IgG fusion protein, which is known to inhibit the activity of extracellular BDNF, and to K252a, a tyrosine kinase inhibitor. Both compounds blocked the NMDA-mediated TrkB tyrosine phosphorylation and subsequently its neuroprotective properties. We suggest that NMDA activates the TrkB receptor via a BDNF autocrine loop, resulting in neuronal survival.

Basic and acidic fibroblast growth factors protect spinal motor neurones in vivo after experimental spinal cord injury.

We studied the effect of a single focal injection of recombinant basic (FGF2) or acidic (FGF1) fibroblast growth factor on the survival of spinal motor neurones at 24 h after a standardized spinal cord contusion injury (SCI) in the rat. Both FGF2 and FGF1 (3 microg), microinjected into the injury site at 5 min post-injury (p.i.), protected at least two functionally important classes of spinal motor neurones, autonomic preganglionic neurones in the intermediolateral (IML) column and somatic motor neurones in the ventral horn (VH). Moreover, there was enhanced choline acetyltransferase (ChAT) immunoreactivity in surviving VH and IML neurones, suggesting an improved functional status. Thus, neurotrophic factors such as FGF2 and FGF1 may contribute to an overall strategy to treat acute SCI and improve recovery of function.

beta-adrenergic receptor-induced activation of nerve growth factor gene transcription in rat cerebral cortex involves CCAAT/enhancer-binding protein delta.

Stimulation of beta-adrenergic receptors (BAR) by clenbuterol (CLE) increases nerve growth factor (NGF) biosynthesis in the rat cerebral cortex but not in other regions of the brain. We have explored the transcription mechanisms that may account for the cortex-specific activation of the NGF gene. Although the NGF promoter contains an AP-1 element, AP-1-binding activity in the cerebral cortex was not induced by CLE, suggesting that other transcription factors govern the brain area-specific induction of NGF. Because BAR activation increases cAMP levels, we examined the role of CCAAT/enhancer-binding proteins (C/EBP), some of which are known to be cAMP-inducible. In C6-2B glioma cells, whose NGF expression is induced by BAR agonists, (i) CLE increased C/EBPdelta-binding activity, (ii) NGF mRNA levels were increased by overexpressing C/EBPdelta, and (iii) C/EBPdelta increased the activity of an NGF promoter-reporter construct. Moreover, DNase footprinting and deletion analyses identified a C/EBPdelta site in the proximal region of the NGF promoter. C/EBPdelta appears to be responsible for the BAR-mediated activation of the NGF gene in vivo, since CLE elicited a time-dependent increase in C/EBPdelta-binding activity in the cerebral cortex only. Our data suggest that, while AP-1 may regulate basal levels of NGF expression, C/EBPdelta is a critical component determining the area-specific expression of NGF in response to BAR stimulation.

Brain-derived neurotrophic factor and basic fibroblast growth factor downregulate NMDA receptor function in cerebellar granule cells.

Evidence has accumulated to suggest that the NMDA glutamate receptor subtype plays an important role in neuronal degeneration evoked by hypoxia, ischemia, or trauma. Cerebellar granule cells in culture are vulnerable to NMDA-induced neuronal excitotoxicity. In these cells, brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (FGF2) prevent the excitotoxic effect of NMDA. However, little is known about the molecular mechanisms underlying the protective properties of these trophic factors. Using cultured rat cerebellar granule cells, we investigated whether BDNF and FGF2 prevent NMDA toxicity by downregulating NMDA receptor function. Western blot and RNase protection analyses were used to determine the expression of the various NMDA receptor subunits (NR1, NR2A, NR2B, and NR2C) after BDNF or FGF2 treatment. FGF2 and BDNF elicited a time-dependent decrease in the expression of NR2A and NR2C subunits. Because NMDA receptor activation leads to increased intracellular Ca2+ concentration ([Ca2+]i), we studied the effect of the BDNF- and FGF2-induced reduction in NR2A and NR2C synthesis on the NMDA-evoked Ca2+ responses by single-cell fura-2 fluorescence ratio imaging. BDNF and FGF2 reduced the NMDA-mediated [Ca2+]i increase with a time dependency that correlates with their ability to decrease NR2A and NR2C subunit expression, suggesting that these trophic factors also induce a functional downregulation of the NMDA receptor. Because sustained [Ca2+]i is believed to be causally related to neuronal injury, we suggest that BDNF and FGF2 may protect cerebellar granule cells against excitotoxicity by altering the NMDA receptor-Ca2+ signaling via a downregulation of NMDA receptor subunit expression.

TrkA receptors delay C6-2B glioma cell growth in rat striatum.

Nerve growth factor (NGF) acts as an anti-mitogenic factor in C6-2B glioma cells stably expressing TrkA (C6trk+). To study the effect of TrkA on cell growth in vivo, we grafted mock and C6trk+ cells into the striatum of ACI nude rats. Thy 1.1 and p75NTR immunohistochemistry revealed that wild type C6-2B cells formed a tumor mass in the striatum by 14 days. In contrast, C6trk+ transplanted rats did not show the presence of a significant tumor mass until 71 days. Analysis of this tumor showed that expression of TrkA was retained, but the synthesis of NGF was abolished. Our data encourage the speculation that expression of TrkA in glioblastoma in vivo will attenuate tumor progression.

Differential induction of nerve growth factor and basic fibroblast growth factor mRNA in neonatal and aged rat brain.

Stimulation of glucocorticoid or beta-adrenergic receptors (BAR) has been shown to increase nerve growth factor (NGF) biosynthesis in adult rat brain. Little is known about the role of these receptors in the regulation of NGF expression in neonatal and aged brain. We have examined the effect of the synthetic glucocorticoid dexamethasone (DEX) and the BAR agonist clenbuterol (CLE) on the levels of NGF mRNA in neonatal (8 day old), adult (3 month old) and aged (24 month old) rats. By 3 h, DEX (0.5 mg/kg, s.c.) evoked a comparable increase in NGF mRNA in the cerebral cortex and hippocampus in both 8-day and 3-month-old rats. In contrast, CLE (10 mg/kg, i.p.) failed to change NGF mRNA levels in neonatal rats, while increasing (2-3-fold) NGF mRNA levels in the cerebral cortex of adult rats. In 24-month-old rats, both DEX and CLE elicited only a modest increase in NGF mRNA. This increase was, however, anatomically and temporally similar to that observed in adult animals. The weak effect of DEX or CLE was not related to a down-regulation of receptor function because both DEX and CLE were able to elicit a comparable increase in the mRNA levels for basic fibroblast growth factor (FGF2) in neonatal, adult and aged rat brain. Our data demonstrate that induction of NGF expression by neurotransmitter/hormone receptor activation varies throughout life and suggest that pharmacological agents might be useful tools to enhance trophic support in aging.

2,3-Dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX) increases fibroblast growth factor mRNA levels after contusive spinal cord injury.

We have previously demonstrated that the glutamatergic receptor (AMPA) antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo(F)-quinoxaline (NBQX) reduces functional deficits in a standardized rat model of contusive spinal cord injury (SCI). NBQX not only acted to protect neurons from excitotoxicity but also, unexpectedly, enhanced sparing of white matter including axons of descending pathways. We have therefore investigated mechanisms through which NBQX could produce beneficial effects for white matter. We report here that NBQX elicits a rapid and selective induction of FGF2 mRNA levels in injured spinal cord. This novel effect could contribute to the therapeutic properties of NBQX in the treatment of SCI.

Toward the development of strategies to prevent ischemic neuronal injury. In vitro studies.

Cerebellar granule cells in culture, which are extremely vulnerable to excitotoxin glutamate or N-methyl-D-aspartate (NMDA), were used to study mechanisms of neuronal cell death and protection. Paradoxically, pretreatment of these cells with subtoxic concentrations of NMDA markedly blocked the neurotoxicity resulting from subsequent exposure to glutamate or NMDA. The NMDA-mediated neuroprotection can be antagonized by pretreatment of these cells with protein synthesis inhibitors, suggesting an involvement of protein(s) with neuroprotectant properties, most likely neurotrophic factors. Because basic fibroblast growth factor (BFGF) is well known to prevent neuronal cell death following mechanical or chemical injury, we have tested whether NMDA increases the synthesis of bFGF in cerebellar granule cells. NMDA elicited a rapid and time-dependent increase in bFGF mRNA, suggesting that availability of this trophic factor may play a role in the NMDA-mediated neuroprotection.

Engraftment of C6-2B cells into the striatum of ACI nude rats as a tool for comparison of the in vitro and in vivo phenotype of a glioma cell line.

The C6-2B is a well-characterized glioma cell line used extensively in the study of malignant glial biology. While the C6-2B cell line has traditionally been thought of as a homogenous cell line, the in vitro phenotype of the C6-2B cell line can vary considerably depending on the culture technique used and the stratum on which the cells are grown. Thus, we asked whether the in vitro phenotype of the C6-2B cell line was significantly different than the in vivo phenotype of the cell line once it was engrafted into the striatum of nude rats. Under culture conditions used in our laboratory, 100% of the C6 cells were found to express p75, the low-affinity nerve growth factor (NGF) receptor, and Major Histocompatability Class I (MHC Class I), while only 10-15% demonstrated vimentin reactivity. Immunohistochemistry was consistently negative for GFAP, trkA (the high-affinity receptor for NGF), CD4, CD8, and a macrophage specific marker (Ox-41). Once engrafted into the striatum of nude rats, the cells remained 100% p75 and MHC Class I positive, and again, only 15% of the cells demonstrated vimentin reactivity. The grafted cells retained this characteristic for 28 days in vivo. Although an immunoincompetent host was selected to minimize the effects an inflammatory response would have on the graft, a transient inflammatory response was detected. During the first week of engraftment, numerous MHC class II cells, some of which were macrophages, were seen infiltrating the graft. However, by 4 weeks postengraftment, no inflammatory cells were appreciated in the graft and surprisingly little reactive gliosis was seen in the penumbra of the tumor mass. Thus, the limited number of in vitro phenotypic characteristics we examined in the C6-2B cell line remained constant once the cells were engrafted into the striatum of athymic nude rats.

Biological activity of interleukin-10 in the central nervous system.

Cytokines play a crucial role as mediators of inflammation. Astrocytes and microglia are the two major glial cells involved in the central nervous system immune responses. In this study we examined the effects of interleukin-10 (IL-10), one of the naturally occurring inhibitory cytokines, on different types of glial cells in culture such as rat astrocytes, hamster microglia and C6-2B glioma cells. Phosphorylation of signal transducers and activators of transcription (STAT) proteins was used as a marker for IL-10 activity. Within minutes, IL-10 elicited a strong and weak increase in STAT3 and STAT1 phosphorylation, respectively, in human T lymphocytes, suggesting that STAT3 is a main IL-10 signaling event in these cells. In contrast, IL-10 failed to induce STAT3 in glial cells, but elicited a weak increase in STAT1 phosphorylation in microglia and C6-2B glioma cells only, suggesting that in some glial cell population(s) IL-10 may produce cellular responses via activation of the STAT1 pathway. Moreover, in C6-2B cells, IL-10 elicited a decrease in the level of basic fibroblast growth factor mRNA. A similar decrease was observed in adult rat hypothalamus, indicating that this cytokine may regulate glial production of trophic factors. Our data suggest that IL-10 may play a role in glial cell differentiation and proliferation.

Increased basic fibroblast growth factor expression following contusive spinal cord injury.

Neurotrophic factors appear to be crucial for the survival and potential regeneration of injured neurons. We have previously demonstrated that contusive spinal cord injury (SCI) increases the levels of mRNA for basic fibroblast growth factor (FGF2). To determine whether FGF2 protein levels also increase, Western blot analysis was performed on extracts of spinal cord tissue after a standardized SCI and compared to laminectomy controls. In spinal cord extracts, a monoclonal antibody to FGF2 recognized various molecular forms of FGF2 (18-24 kDa) and some characteristic proteolytic fragments. Extracts of spinal cords 1 day after SCI showed a slight increase in the levels of these polypeptides. By 4 days, a significant increase (two-fold) was detected in the levels of the 18-kDa and higher molecular weight forms as well as the proteolytic fragments. Immunohistochemical analyses on spinal cord tissue sections confirmed an increased cellular (glial) FGF2 as well as interstitial immunoreactivity surrounding neurons and along blood vessels. Heparinpurified spinal cord extracts from tissue 4 days after SCI showed increased biological activity as indicated by their ability to (i) increase [3H]thymidine incorporation in cultures of Balb/c 3T3 cells and (ii) induce phosphorylation of suc-associated neurotrophic factor-induced tyrosine-phosphorylated target, a FGF2 target protein. These data suggest that SCI induces increased FGF2 expression and support the hypothesis that FGF2 may play a role in the partial recovery of function seen following SCI.